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Global Analysis of Protein Stability by Temperature and Chemical Denaturation

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Global Analysis of Protein Stability by Temperature and Chemical Denaturation bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Global analysis of protein stability by temperature and chemical denaturation Louise Hamborg, Emma Wenzel Horsted, Kristoffer Enøe Johansson, Martin Willemoës, Kresten Lindorff-Larsen and Kaare Teilum* Structural Biology and NMR laboratory and the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, 2200 Copenhagen N, Denmark * Corresponding author Phone: +45 35 32 20 29 Postal Address: Ole Maaloes Vej 5, 2200 Copenhagen N, Denmark Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5 – 1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 µl is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 µg of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook. Keywords Protein stability, chemical denaturation, temperature denaturation, data analysis. Abbreviations ACBP, bovine acyl-coenzyme A binding protein; ASA, accessible surface area; CI2, barley chymotrypsin inhibitor 2; DSC, differential scanning calorimetry; LEM, linear extrapolation model. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Introduction The stability of the native state of a protein determines under which conditions the protein is folded and thus active. Accurate measurements of protein stability are important if we wish to understand the underlying interactions that stabilizes a protein structure and manipulate proteins to be more (or less) stable. Protein stabilities are typically determined by gradually changing temperature or the concentration of a chemical denaturant and measuring the unfolding by a spectroscopic technique or calorimetry. For highly stable proteins, e.g. from thermophilic organisms, proteins may not necessarily unfold at temperatures below 100 °C and it may be thus be necessary to include a chemical denaturant to reach the unfolded state. Similarly, some proteins need elevated temperatures to completely unfold in a chemical denaturant. Protein denaturation may be measured by differential scanning calorimetry (DSC), where the excess heat capacity of unfolding is quantified, and gives a direct measure of the enthalpy for folding and the melting temperature (Tm) [1]. DSC thus provides a full thermodynamic description of the folding process as long as it is reversible, though fitting DSC experiments and determining the thermodynamic stability at ambient temperatures is not always trivial. Protein folding can also be followed by different types of spectroscopies. Most widely applied are fluorescence spectroscopy and CD spectroscopy [2]. In fluorescence spectroscopy, the process may either be probed by the change in the intrinsic protein fluorescence from tryptophan or tyrosine residues or by the change in fluorescence from extrinsic fluorescent probes such as SYPRO orange and ANS that binds to hydrophobic parts of the protein [3]. The fluorescence signals of fluorophores are highly dependent on the surrounding environment, which can be used as a measure for protein unfolding [4]. The Prometheus nanoDSF apparatus (NanoTemper Technologies GmbH) makes it possible to measure unfolding using small volumes (10 µl) and low concentrations of proteins (down to 5 µg/mL) and allows for the analysis of up to 48 samples simultaneously. The instrument uses quartz capillaries as sample cuvettes and measures the intrinsic protein fluorescence at 330 nm and 350 nm (with excitation at 280 nm) as a function of temperature from 15 °C to 95 °C. It is thus possible to perform unfolding experiments by temperature and denaturant on small amounts of protein. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Here we have performed two-dimensional unfolding experiments on bovine acyl-coenzyme A binding protein (ACBP) and barley chymotrypsin inhibitor 2 (CI2). The folding mechanisms of these proteins have been intensively studied [5,6,15–18,7–14] and ACBP and CI2 thus serve as an excellent set of model proteins for validating our method. By fitting temperature denaturation profiles at several denaturant concentrations to a multivariate function with both temperature and denaturant concentration as independent variables it is possible to estimate ∆Hm, ∆Cp, Tm and the m-values as demonstrated by others [19]. We show that the total number of different GuHCl concentrations needed for a robust parameter estimate can be drastically reduced, and that both Tm and ∆H at 298K still get reliably determined from only five samples for both ACBP and CI2. With the need of only a low amount of protein for each sample and the few samples, the method is well suited if the available amount of protein is limited, and for measuring many variants in parallel. Theory Temperature denaturation In the following, we will consider the denaturation of a protein that (un-)folds reversibly by a two-state process (� ⇌ �). The equilibrium constant K for unfolding is then: � = [�]/[�]. K will depend on the temperature T according to: ∆#!(%) ! (1) �(�) = � '% where DG0(T) is the temperature dependent change in Gibbs free energy for unfolding and R is the gas constant. The temperature midpoint of the unfolding, Tm, defines the temperature at which the fractions 0 0 of native and denatured protein are equal (∆G (Tm) = 0). DG (T) is given by: ( ( � ( � (2) ∆� (�) = ∆�) 11 − 4 + ∆�* 1� − �) − � ∙ ln 1 44 �) �) 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ( where ∆Hm is the change in enthalpy upon unfolding at Tm and ∆�* is the change in heat capacity of the system which here is assumed to be independent of temperature [20]. For a two-state process, the fluorescence signal (or any other spectroscopic signal) of a thermally induced denaturation can be described by the equilibrium constant and the temperature dependent fluorescence signals of the native and denatured protein, fN(T) and fD(T), respectively. Also, for a two- state unfolding process we have that the populations of the two states add to 1, pN + pD = 1. Combining this and using equation 1 we get: � (�) � (�) ∙ �(�) �(�) = � (�)� (�) + � (�)� (�) = + + , + + , , 1 + �(�) 1 + �(�) (3) ∆#! -! . � (�) + � (�) ∙ � '% = + , ∆#! -! . 1 + � '% The temperature dependence of the fluorescence signals of the native state is often linear whereas that of the denatured state is curved [21] and often may be described by adding a quadratic term [22], though other non-linear functions have also been used [23]. Thus we have: �+(�) = �+(�/01) + �( ∙ ∆� (4) 3 �,(�) = �,(�/01) + �2 ∙ ∆� + �2 ∙ ∆� where, a0, a1 and b1 are constants describing the temperature dependence of the fluorescence signal. ∆� is defined as ∆� = � − �/01, where Tref is an arbitrary chosen reference temperature for the fluorescence signal. Introducing the temperature dependence of the fluorescence signals and expanding DG according to equation 2 gives: 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. 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